Novel vectors which are replication competent in neoplastic cells and which overexpress an adenovirus death protein are disclosed. Some of the disclosed vectors are replication-restricted to neoplastic cells or to neoplastic alveolar type II cells. compositions and methods for promoting the death of neoplastic cells using these replication-competent vectors are also disclosed.
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1. A recombinant adenovirus vector which is replication-competent in a neoplastic cell and which overexpresses an adenovirus death protein (ADP), wherein said overexpression is defined as overexpression relative to dl309.
10. A method for promoting death of a neoplastic cell in a patient comprising contacting the neoplastic cell with at least one adenoviral vector which is replication competent in the neoplastic cell and which overexpresses an adenovirus death protein (ADP), wherein said overexpression is defined as overexpression relative to dl309.
18. A composition comprising:
a first recombinant adenovirus which is replication competent in a neoplastic cell and overexpresses an adenovirus death protein (ADP), wherein said overexpression is defined as overexpression relative to dl309; and
a second recombinant adenovirus which is replication defective and which expresses an anti-cancer gene product,
wherein the first recombinant adenovirus complements replication of the second recombinant adenovirus.
2. The recombinant adenoviral vector of
4. The recombinant adenoviral vector of
6. The recombinant adenoviral vector of
8. The recombinant adenoviral vector of
9. The recombinant adenoviral vector of
11. The method of
12. The method of
13. The method of
14. The method of
15. The method of
16. The method of
17. The method of
19. The composition of
20. The composition of
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The present application is a continuation of application Ser. No. 11/249,873, filed Oct. 13, 2005, now U.S. Pat. No. 7,608,255 which is a continuation of U.S. Ser. No. 09/351,778, filed Jul. 12, 1999, now U.S. Pat. No. 7,589,069, the entire contents of each of which are incorporated herein by reference in their entirety.
This invention was made with government support under a grant from the National Institutes of Health, Grant Number RO1 CA71704. The government has certain rights in this invention.
(1) Field of the Invention
This invention relates generally to the treatment of cancer and more particularly to vectors which replicate in neoplastic cells and which overexpress an adenovirus death protein (ADP) and to the use of these vectors in treating human cancer.
(2) Description of the Related Art
Cancer is a leading cause of death in the United States and elsewhere. Depending on the type of cancer, it is typically treated with surgery, chemotherapy, and/or radiation. These treatments often fail: surgery may not remove all the cancer; some cancers are resistant to chemotherapy and radiation therapy; and chemotherapy-resistant tumors frequently develop. New therapies are necessary, to be used alone or in combination with classical techniques.
One potential therapy under active investigation is treating tumors with recombinant viral vectors expressing anti-cancer therapeutic proteins. Adenovirus-based vectors contain several characteristics that make them conceptually appealing for use in treating cancer, as well as for therapy of genetic disorders. Adenoviruses (hereinafter used interchangeably with “Ads”) can easily be grown in culture to high titer stocks that are stable. They have a broad host range, replicating in most human cancer cell types. Their genome can be manipulated by site-directed mutation and insertion of foreign genes expressed from foreign promoters.
The adenovirion consists of a DNA-protein core within a protein capsid (reviewed by Stewart et al., “Adenovirus structure by x-ray crystallography and electron microscopy.” in: The Molecular Repertoire of Adenoviruses, Doerfler, W. et al., (ed)., Springer-Verlag, Heidelberg, Germany, p. 25-38). Virions bind to a specific cellular receptor, are endocytosed, and the genome is extruded from endosomes and transported to the nucleus. The genome is a linear duplex DNA of about 36 kbp, encoding about 36 genes (
The Ad vectors being investigated for use in anti-cancer and gene therapy are based on recombinant Ad's that are either replication-defective or replication-competent. Typical replication-defective Ad vectors lack the E1A and E1B genes (collectively known as E1) and contain in their place an expression cassette consisting of a promoter and pre-mRNA processing signals which drive expression of a foreign gene. These vectors are unable to replicate because they lack the E1A genes required to induce Ad gene expression and DNA replication. In addition, the E3 genes are usually deleted because they are not essential for virus replication in cultured cells.
A number of investigators have constructed replication-defective Ad vectors expressing anti-cancer therapeutic proteins. Usually, these vectors have been tested by direct injection of human tumors growing in mouse models. Most commonly, these vectors express the thymidine kinase gene from herpes simplex virus, and the mice are treated with gancyclovir to kill cells transduced by the vector (see e.g., Felzmann et al., Gene Ther. 4:1322-1329, 1997). Another suicide gene therapy approach involves injecting tumors with a replication defective Ad vector expressing cytosine deaminase, followed by administration of 5-fluorocytosine (Topf et al., Gene Ther. 5::507-513, 1998). Investigators have also prepared and tested replication-defective Ad vectors expressing a cytokine-such as IL-2, IL-12, IL-6, tumor necrosis factor (TNF), type I interferons, or the co-stimulatory molecule B7-1 in the anticipation that the Ad-expressed cytokine will stimulate an immune response, including cytotoxic T-lymphocytes (CTL), against the tumor (Felzmann et al., supra; Putzer et al., Proc. Natl. Acad. Sci. USA 94:10889-10894, 1997). Other vectors express tumor antigens (e.g. melanoma MART1), proteins that de-regulate the cell cycle and induce apoptosis (p53, pRB, p21Kip1/WAF1, p16CDKN2, and even Ad E1A), and ribozymes. An Ad vector expressing FasL induces apoptosis and tumor regression of a mouse tumor (Arai et al., Proc. Natl. Acad. Sci. USA 94:13862-13867, 1997).
Despite these generally positive reports, it is recognized in the art that replication-defective Ad vectors have several characteristics that make them suboptimal for use in therapy. For example, production of replication-defective vectors requires that they be grown on a complementing cell line that provides the E1A proteins in trans. Such cell lines are fastidious, and generation of virus stocks is time-consuming and expensive. In addition, although many foreign proteins have been expressed from such vectors, the level of expression is low compared to Ad late proteins.
To address these problems, several groups have proposed using replication-competent Ad vectors for therapeutic use. Replication-competent vectors retain Ad genes essential for replication and thus do not require complementing cell lines to replicate. Replication-competent Ad vectors lyse cells as a natural part of the life cycle of the vector. Another advantage of replication-competent Ad vectors occurs when the vector is engineered to encode and express a foreign protein. Such vectors would be expected to greatly amplify synthesis of the encoded protein in vivo as the vector replicates. For use as anti-cancer agents, replication-competent viral vectors would theoretically also be advantageous in that they should replicate and spread throughout the tumor, not just in the initial infected cells as is the case with replication-defective vectors.
Wyeth Laboratories developed replication-competent Ad vectors for vaccination purposes, using vaccine strains of Ad serotypes 4, 7, and 5 (Lubeck et al., AIDS Res. Hum. Retroviruses 10:1443-1449, 1994). Foreign genes were inserted into the E3 region (with the E3 genes deleted) or into a site at the right end of the genome. Two foreign genes used were hepatitis B surface antigen and the HIV envelope protein. They obtained good expression in culture, and were able to raise antisera in animal models. Phase I human trials were ambiguous, and the project was mostly abandoned.
Onyx Pharmaceuticals recently reported on adenovirus-based anti-cancer vectors which are replication deficient in non-neoplastic cells but which exhibit a replication phenotype in neoplastic cells lacking functional p53 and/or retinoblastoma (pRB) tumor suppressor proteins (U.S. Pat. No. 5,677,178; Heise et al., Nature Med. 6:639-645, 1997; Bischoff et al., Science 274:373-376, 1996). This phenotype is reportedly accomplished by using recombinant adenoviruses containing a mutation in the E1B region that make the encoded E1B-55K protein incapable of binding to p53 and/or a mutation(s) in the E1A region which make the encoded E1A protein (p289R or p243R) incapable of binding to pRB and/or the cellular 300 kD polypeptide and/or the 107 kD polypeptide. E1B-55K has at least two independent functions: it binds and inactivates the tumor suppressor protein p53, and it is required for efficient transport of Ad MRNA from the nucleus. Because these E1B and E1A viral proteins are involved in forcing cells into S-phase, which is required for replication of adenovirus DNA, and because the p53 and pRB proteins block cell cycle progression, the recombinant adenovirus vectors described by Onyx should replicate in cells defective in p53 and/or pRB, which is the case for many cancer cells, but not in cells with wild-type p53 and/or pRB. Onyx has reported that replication of an adenovirus lacking E1B-55K, which is named ONYX-015, was restricted to p53-minus cancer cell lines (Bischoffet al., supra), and that ONYX-015 slowed the growth or caused regression of a p53-minus human tumor growing in nude mice (Heise et al., supra). Others have challenged the Onyx report claiming that replication of ONYX-015 is independent of p53 genotype and occurs efficiently in some primary cultured human cells (Harada and Berk, J. Virol 73:5333-5344, 1999). ONYX-015 does not replicate as well as wild-type adenovirus because E1B-55K is not available to facilitate viral mRNA transport from the nucleus. Also, ONYX-015 expresses less ADP than wild-type virus (see Example 1 below). As an extension of the ONYX-015 concept, a replication-competent adenovirus vector was designed that has the gene for E1B-55K replaced with the herpes simplex virus thymidine kinase gene (Wilder et al., Gene Therapy 6:57-62, 1999). The group that constructed this vector reported that the combination of the vector plus gancyclovir showed a therapeutic effect on a human colon cancer in a nude mouse model (Wilder et al., Cancer Res. 59:410-413, 1999). However, this vector lacks the gene for ADP, and accordingly, the vector will lyse cells and spread from cell-to-cell less efficiently than an equivalent vector that expresses ADP. The gene for ADP is also lacking in another replication-competent adenovirus vector that has been described, in which a minimal enhancer/promoter of the human prostate specific antigen was inserted into the adenovirus E1A enhancer/promoter (Rodriguez et al., Cancer Res. 57:2559-2563, 1997).
Thus, there is a continuing need for vectors that replicate and spread efficiently in tumors but that can be modified such that they replicate poorly or not at all in normal tissue.
Briefly, therefore, the present invention is directed to novel vectors which are replication competent in neoplastic cells and which overexpress an adenovirus death protein (ADP). The work reported herein demonstrates the discovery that overexpression of ADP by a recombinant adenovirus allows the construction of a replication-competent adenovirus that kills neoplastic cells and spreads from cell-to-cell at a rate similar to or faster than that exhibited by adenoviruses expressing wild-type levels of ADP, even when the recombinant adenovirus contains a mutation that would otherwise reduce its replication rate in non-neoplastic cells. This discovery was unexpected because it could not have been predicted from what was known about adenovirus biology that Ad vectors overexpressing ADP remain viable and that the infected cells are not killed by the higher amounts of ADP before the Ad vector produces new virus particles that can spread to other tumor cells. Indeed, naturally-occurring adenoviruses express ADP in low amounts from the E3 promoter at early stages of infection, and begin to make ADP in large amounts only at 24-30 h p.i., once virions have been assembled in the cell nucleus. It is believed that other non-adenoviral vectors can be used to deliver ADP's cell-killing activity to neoplastic cells, including other viral vectors and plasmid expression vectors.
Thus, in one preferred embodiment, the ADP-expressing vector comprises a recombinant adenovirus lacking expression of at least one E3 protein selected from the group consisting of: gp19K; RIDα (also known as 10.4K); RIDβ (also known as 14.5K) and 14.7K. Because these E3 proteins inhibit immune-mediated inflammation and/or apoptosis of Ad-infected cells, it is believed that a recombinant adenovirus lacking one or more of these E3 proteins will stimulate infiltration of inflammatory and immune cells into a tumor treated with the adenovirus and that this host immune response will aid in destruction of the tumor as well as tumors that have metastasized. The ADP expressed by preferred embodiments comprises a naturally-occurring amino acid sequence from a human adenovirus of subgroup C, namely Ad1, Ad2, Ad5 and Ad6.
In another embodiment, replication of the vector is restricted to neoplastic cells. Such replication-restricted vectors are useful in treating cancer patients in which it is desirable to eliminate or reduce damage to normal cells and tissues that might be caused by the vector, particularly viral vectors that kill the host cell as part of their life cycle. In preferred embodiments, a recombinant adenovirus has a replication-restricted phenotype because the recombinant adenovirus is incapable of expressing an E1A viral protein which binds the pRB and the p300/CBP proteins or because the E4 promoter has been substituted with a promoter that is activated only in neoplastic cells.
In yet another embodiment, the invention provides a vector which overexpresses ADP and whose replication is under the control of a tissue specific promoter or an inducible promoter. In preferred embodiments, the vector comprises a recombinant adenovirus in which the tissue specific promoter or inducible promoter is substituted for the E4 promoter. Such vectors are useful for restricting replication of the vector and its ADP-mediated cell killing to cells of a particular type or to cells exposed to an exogenous agent that activates the promoter. A preferred tissue-specific or inducible vector also expresses a phenotype that restricts its replication to neoplastic cells.
In yet another embodiment, the invention provides a vector which overexpresses ADP but which is not restricted to tumors by a specific genetic modification. Such a vector is more destructive to neoplastic cells than even the naturally occurring Ad's of subgroup C. In preferred embodiments, this vector could be used for patients with terminal cancer not treatable by another method, and who have pre-existing neutralizing antibodies to Ad or to which neutralizing antibodies can be administered.
In still another embodiment, the invention provides a composition comprising a first recombinant virus which is replication competent in a neoplastic cell and overexpresses the adenovirus death protein. In one embodiment, the recombinant virus is contained within a delivery vehicle comprising a targeting moiety that limits delivery of the virus to cells of a certain type. With this embodiment, the replication-competent vector can be of any ADP-overexpressing configuration described herein. In some embodiments, the composition also comprises a second recombinant virus which is replication-defective and which expresses an anti-cancer gene product. The recombinant virus complements spread of the replication-defective virus, as well as its encoded anti-cancer product, throughout a tumor. In preferred embodiments, the first recombinant virus is a recombinant adenovirus whose replication is restricted to neoplastic cells and/or which lacks expression of one or more of the E3 gp19K; RIDα; RIDβ; and 14.7K proteins.
The ADP-expressing vectors and compositions of the invention are useful in a method for promoting death of a neoplastic cell. The method comprises contacting the neoplastic cell with a vector which is replication-competent in the neoplastic cell and which overexpresses ADP. Where the neoplastic cell comprises a tumor in a patient, the vector is administered directly to the tumor or, in other embodiments, the vector is administered to the patient systemically or in a delivery vehicle containing a targeting moiety that directs delivery of the vector to the tumor. In embodiments where the vector is a recombinant virus, the method can also comprise passively immunizing the patient against the virus.
In yet another embodiment of the invention, the vector may be used in combination with radiation therapy. The radiation therapy can be any form of radiation therapy used in the art such as for example, external beam radiation such as x-ray treatment, radiation delivered by insertion of radioactive materials within the body near or at the tumor site such as treatment with gamma ray emitting radionuclides, particle beam therapy which utilizes neutrons or charged particles and the like. In addition, this embodiment encompasses the use of more than one of the vectors of the present invention in a cocktail in combination with radiation therapy.
Another embodiment of the invention involves the use of the recombinant vector in combination with chemotherapy as has been disclosed for other adenovirus vectors (U.S. Pat. No. 5,846,945). Chemotherapeutic agents are known in the art and include antimetabolites including pyrimidine-analogue and purine-analogue antimetabolies, plant alkaloids, antitumor antibiotics, alkylating agents and the like. The use of more than one of the vectors of the present invention with a chemotherapeutic agent or agents is also contemplated within this embodiment.
Among the several advantages found to be achieved by the present invention, therefore, may be noted the provision of replication-competent vectors, particularly viruses, which rapidly kill cancer cells and spread from cell-to-cell in a tumor; the provision of such vectors whose replication can be induced or which is restricted to tumors and/or to cells of a certain tissue type; and the provision of compositions and methods for anti-cancer therapy which cause little to no side effects in normal tissues.
In accordance with the present invention, it has been discovered that overexpression of ADP by a recombinant adenovirus results in faster lysis of cells and spread of the virus throughout a cell monolayer than viruses expressing wild-type levels of ADP. It has also been discovered that this function for ADP is manifest in an adenovirus which contains E1A mutations that restrict adenoviral replication to neoplastic cells. Thus, vectors which are both replication competent in neoplastic cells and which overexpress ADP should be useful in anti-cancer therapy.
In the context of this disclosure, the following terms will be defined as follows unless otherwise indicated:
“Naturally-occurring” as applied to an object such as a polynucleotide, polypeptide, or virus means that the object can be isolated from a source in nature and has not been intentionally modified by a human.
“Neoplastic cell” means a cell which exhibits an aberrant growth phenotype characterized by a significant loss of control of cell proliferation and includes actively replicating cells as well as cells in a temporary non-replicative resting state (G1 or G2). A neoplastic cell may have a well-differentiated phenotype or a poorly-differentiated phenotype and may comprise a benign neoplasm or a malignant neoplasm.
“Recombinant virus” means any viral genome or virion which is different than a wild-type virus due to a deletion, insertion, or substitution of one or more nucleotides in the wild-type viral genome. The recombinant virus can have changes in the number of amino acid sequences encoded and expressed or in the amount or activity of proteins expressed by the virus. In particular, the term includes recombinant viruses generated by the intervention of a human.
“Replication-competent” as applied to a vector means that the vector is capable of replicating in normal and/or neoplastic cells. As applied to a recombinant virus, “replication-competent” means that the virus exhibits the following phenotypic characteristics in normal and/or neoplastic cells: cell infection; replication of the viral genome; and production and release of new virus particles; although one or more of these characteristics need not occur at the same rate as they occur in the same cell type infected by a wild-type virus, and may occur at a faster or slower rate. Where the recombinant virus is derived from a virus such as adenovirus that lyses the cell as part of its life cycle, it is preferred that at least 5 to 25% of the cells in a cell culture monolayer are dead 5 days after infection. Preferably, a replication-competent virus infects and lyses at least 25 to 50%, more preferably at least 75%, and most preferably at least 90% of the cells of the monolayer by 5 days post infection (p.i.).
“Replication-defective” as applied to a recombinant virus means the virus is incapable of or is greatly compromised in, replicating its genome in any cell type in the absence of a complementing replication-competent virus. Exceptions to this are cell lines such as 293 cells that have been engineered to express adenovirus E1A and E1B proteins.
“Replication-restricted” as applied to a vector of the invention means the vector replicates better in a dividing cell, i.e. either a neoplastic cell or a non-neoplastic, dividing cell, than in a cell of the same type that is not neoplastic and/or not dividing, which is also referenced herein as a normal, non-dividing cell. Preferably, a replication-restricted virus kills at least 10% more neoplastic cells than normal, non-dividing cells in cell culture monolayers of the same size, as measured by the number of cells showing cytopathic effects (CPE) at 5 days p.i. More preferably, between 25% and 50%, and even more preferably, between 50% and 75% more neoplastic than normal cells are killed by a replication-restricted virus. Most preferably, a replication-restricted adenovirus kills between 75% and 100% more neoplastic than normal cells in equal sized monolayers by 5 days p.i.
In one embodiment the invention provides a vector that is replication-competent in neoplastic cells and which overexpresses an ADP. Vectors useful in the invention include but are not limited to plasmid-expression vectors, bacterial vectors such as Salmonella species that are able to invade and survive in a number of different cell types, vectors derived from DNA viruses such as human and non-human adenoviruses, adenovirus associated viruses (AAVs), poxviruses, herpesviruses, and vectors derived from RNA viruses such as retroviruses and alphaviruses. Preferred vectors include recombinant viruses engineered to overexpress an ADP. Recombinant adenoviruses are particularly preferred for use as the vector, especially vectors derived from Ad1, Ad2, Ad5 or Ad6.
Vectors according to the invention overexpress ADP. As applied to recombinant Ad and AAV vectors, the term “overexpresses ADP” means that more ADP molecules are made per viral genome present in a dividing cell infected by the vector than expressed by any previously known recombinant adenoviral vector or AAV in a dividing cell of the same type. As applied to other, non-adenoviral vectors, “overexpresses ADP” means that the virus expresses sufficient ADP to lyse a cell containing the vector.
Vectors overexpressing ADP can be prepared using routine methodology. (See, e.g., A Laboratory Cloning Manual, 2nd Ed., vol. 3, Sambrook et al., eds., Cold Spring Harbor Laboratory Press, 1989). For example, a polynucleotide encoding the ADP can be cloned into a plasmid expression vector known to efficiently express heterologous proteins in mammalian cells. The polynucleotide should also include appropriate termination and polyadenylation signals. Enhancer elements may also be added to the plasmid to increase the amount of ADP expression. Viral vectors overexpressing. ADP can be prepared using similar materials and techniques.
Where the virus is a recombinant adenovirus, overexpression of ADP can be achieved in a multitude of ways. In general, any type of deletion in the E3 region that removes a splice site for any of the E3 mRNAs will lead to overexpression of the mRNA for ADP, inasmuch as more of the E3 pre-mRNA molecules will be processed into the mRNA for ADP. This is exemplified in the KD1, KD3, GZ1 and GZ3 vectors (SEQ ID NOS:1-4) whose construction is described below. Other means of achieving overexpression of ADP in Ad vectors include, but are not limited to: insertion of pre-mRNA splicing and cleavage/polyadenylation signals at sites flanking the gene for ADP; expression of ADP from another promoter, e.g. the human cytomegalovirus promoter, inserted into a variety of sites in the Ad genome; and insertion of the gene for ADP behind the gene for another Ad MRNA, together with a sequence on the 5′ side of the ADP sequence that allows for internal initiation of translation of ADP, e.g. the Ad tripartite leader or a viral internal ribosome initiation sequence.
The ADP expressed by a vector according to the invention is any polypeptide comprising a naturally-occurring full-length ADP amino acid sequence or variant thereof that confers upon a vector expressing the ADP the ability to lyse a cell containing the vector such that replicated copies of the vector are released from the infected cell. A preferred full-length ADP comprises the ADP amino acid sequence encoded by Ad1, Ad2, AdS or Ad6. These naturally-occurring ADP sequences are set forth in SEQ ID NOS:5-8, respectively. ADP variants include fragments and deletion mutants of naturally-occurring adenovirus death proteins, as well as full-length molecules, fragments and deletion mutants containing conservative amino acid substitutions, provided that such variants retain the ability, when expressed by a vector inside a cell, to lyse the cell.
Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. Conservatively substituted amino acids can be grouped according to the chemical properties of their side chains. For example, one grouping of amino acids includes those amino acids having neutral and hydrophobic side chains (A, V, L, I, P, W, F, and M); another grouping is those amino acids having neutral and polar side chains (G, S, T, Y, C, N, and Q); another grouping is those amino acids having basic side chains (K, R, and H); another grouping is those amino acids having acidic side chains (D and E); another grouping is those amino acids having aliphatic side chains (G, A, V, L, and I); another grouping is those amino acids having aliphatic-hydroxyl side chains (S and T); another grouping is those amino acids having amine-containing side chains (N, Q, K, R, and H); another grouping is those amino acids having aromatic side chains (F, Y, and W); and another grouping is those amino acids having sulfur-containing side chains (C and M). Preferred conservative amino acid substitutions groups are: R-K; E-D, Y-F, L-M; V-I, and Q-H.
As used herein, an ADP variant can also include modifications of a naturally-occurring ADP in which one or more amino acids have been inserted, deleted or replaced with a different amino acid or a modified or unusual amino acid, as well as modifications such as glycosylation or phosphorylation of one or more amino acids so long as the ADP variant containing the modified sequence retains cell lysing activity.
As described below, the inventors herein performed a structure-function analysis of ADP which defined specific domains in ADP required to promote cell death. Using this information, when combined with known recombinant DNA and cloning methodology, it is believed the skilled artisan can readily construct ADP variants of a naturally-occurring adenovirus death protein and test them for cell lysing activity. A preferred ADP deletion mutant comprises an ADP amino acid sequence from any of the deletion mutants dl716, dl715, dl714 and dl737, whose ADP sequences are set forth in SEQ ID NOS:9-12, respectively).
Where the vector is derived from a virus, it is preferred that the virus lack expression of one or more viral proteins involved in avoiding host anti-viral defenses such as immune-mediated inflammation and/or apoptosis of infected cells. For example, adenovirus contains a cassette of genes that prevents killing of Ad-infected cells by the immune system (Wold et al., Semin. Virol., 1998 (8:515-523, 1998). The E3-14.7K protein and the E3 RID (Receptor Internalization and Degradation) protein, which is a complex consisting of RIDα and RIDβ, inhibit apoptosis of Ad-infected cells induced by tumor necrosis factor (TNF) and the Fas ligand which are expressed on, or secreted by, activated macrophages, natural killer (NK) cells, and cytotoxic lymphocytes (CTLs) (Tollefson et al., Nature 392:727-730, 1998). The E3-gp19K protein inhibits CTL-killing of infected cells by blocking transport of MHC class I antigens to the cell surface (Wold et al., supra). Thus, it is believed that infection of tumor cells by such viral vectors will stimulate infiltration of inflammatory cells and lymphocytes into the tumor, and will not prevent infected tumor cells from apoptosis induced by cytolytic cells of the immune system, or against apoptosis inducing cytokines. For example, it is known that when mice are infected with Ad mutants lacking the E3 gp19K, RID and 14.7K proteins there is a dramatic increase (as compared to E3-positive Ad) in infiltration of inflammatory cells and lymphocytes into the infected tissue (Sparer et al., J. Virol. 70:2431-2439, 1996). A similar infiltration of tumors infected by an ADP-expressing viral vector of the invention would be expected to further promote destruction of the tumor by adding an immune system attack to the ADP-mediated killing activity. For example, it is believed that the viral infection will stimulate formation of tumor-specific CTL's that can kill neoplastic cells not only in the tumor but also ones that have metastasized. In addition, it is also expected that vector-specific CTL's will be generated which could attack vector-infected cells if the vector spreads away from the tumor into normal cells. Because viral vectors overexpressing ADP will spread rapidly through the tumor, it is believed these immune mechanisms will have little effect on spread of the vector.
Where the vector is a recombinant adenovirus, it is preferred that the adenovirus lack expression of each of the E3 gp19K, RID, and 14.7K proteins. By “lack expression” and “lacking expression” of a protein(s), “it is meant” that the viral genome contains one or more mutations that inactivates expression of a functional protein, i.e., one having all the functions of the wild-type protein. The inactivating mutation includes but is not limited to substitution or deletion of one or more nucleotides in the encoding gene(s) that prevents expression of functional transcripts or that results in transcripts encoding nonfunctional translation products. A particularly preferred way to inactivate expression of the Ad E3 gp19K, RID, and 14.7K proteins is by deleting the E3 region containing the genes encoding these proteins. Preferably, one or both of the E3 genes encoding the E3 6.7K and 12.5K proteins are also deleted because, as discussed in the Examples below, it is believed that deletion of most or all of the E3 genes other than the ADP gene facilitates overexpression of ADP mRNA by reducing competition for splicing of the major late pre-mRNAs. Preferred Ad vectors containing an E3 deletion that overexpress ADP are GZ1 (SEQ ID NO:3) and GZ3 (SEQ ID NO:4), whose construction and properties are described in the Examples below.
The invention also provides ADP-expressing vectors whose replication is restricted to dividing cells. Any means known to provide such a replication-restricted phenotype may be used. For example, WO 96/40238 describes microbes that preferentially invade tumor cells as well as methods for identifying and isolating bacterial promoters that are selectively activated in tumors. It is also contemplated that expression of one or more vector proteins essential for replication can be placed under the control of the promoter for a cellular gene whose expression is known to be upregulated in neoplastic cells. Examples of such genes include but are not limited to: the breast cancer markers mammaglobin (Watson et al., Oncogene 16:817-824, 1998); BRCA1 (Norris et al., J. Biol. Chem. 270:22777-22782, 1995) and her2/neu (Scott et al., J. Biol. Chem. 269:19848-19858, 1994); and prostate specific antigen (U.S. Pat. No. 5,698,443); surfactant protein B for lung alveoli (Yan et al., J. Biol. Chem. 270:24852-24857, 1995); factor VII for liver (Greenberg et al., Proc. Natl. Acad. Sci. USA 92:12347-12351, 1995); and survivin for cancer in general (Li et al., Nature 396:580-584). Where the vector is an adenovirus, it is contemplated that such tumor-specific promoters can be substituted for the E4 promoter. Because E4 gene products are essential for Ad replication, placing their expression under the control of a tumor-specific promoter should restrict replication of the vector to tumor cells in which the promoter is activated.
Another strategy for restricting replication of ADP-expressing Ad vectors to neoplastic cells is exemplified by the KD1 (SEQ ID NO:1), KD2 (SEQ ID NO:13) and KD3 (SEQ ID NO:2) vectors, whose construction and properties are described in the Examples below. This strategy exploits a pre-existing Ad5 mutant in the E1A gene, named dl1101/1107 (Howe et al., Proc. Natl. Acad. Sci., 87:5883-5887, 1990), also referred to herein as dl01/07, and which can only grow well in cancer cells. The role of E1A is to drive cells from the GO and GI phases of the cell cycle into S-phase. This is achieved by two mechanisms, one involving pRB (and family members), and the other involving p300 and the related protein CBP (DePinho, R. A., Nature 391:533-536, 1998). One domain in E1A binds members of the pRB family. pRB normally exists in the cell as a complex with the transcription factor E2F-1 and E2F family members (E2F), tethered via E2F to E2F binding sites in promoters of cells expressed in S-phase. Here, pRB acts as a transcriptional co-repressor. E1A binding to pRB relieves this repression, and causes the release of E2F from pRB/E2F complexes. Free E2F then activates promoters of genes expressed in S-phase, e.g. thymidine kinase, ribonucleotide reductase, etc. Another domain in E1A binds the p300/CBP transcription adaptor protein complex. p300/CBP is a transcriptional co-activator that binds many different transcription factors and accordingly is targeted to promoters. p300/CBP has intrinsic histone acetyltransferase activity. E1A binding to p300/CBP is believed to inhibit this histone acetyltransferase activity, allowing acetylation of histones and repression of transcription (Chakravarti et al., Cell 96:393-403, 1999; Hamamori et al., Cell 96:405-413, 1999). Conceivably, some of the genes that are repressed as a result of E1A interacting with p300/CBP to play a role in blocking the cell cycle, although this is not known. Cancer cells are cycling, so they have free E2F and presumably some p300/CBP-regulated genes are repressed. Consistent with these ideas, E1A must bind both p300/CBP and the pRB family in order to transform primary cells to a constitutively cycling state (Howe et al., supra). The mutant dl01/07 lacks both the p300/CBP- and pRB-binding domains and, as expected, it replicates very poorly in non-dividing “normal” cells or serum-starved cancer cells, but well in growing cancer cells. As described below, the growth of the KD1 and KD3 vectors, which contain the dl01/07 E1A mutation, is very much better in dividing cancer cells as compared to non-dividing cells. Because the dl01/07 mutant is completely defective in oncogenic transformation of rat cells (Howe et la., supra), vectors according to the invention that contain this E1A mutation cannot induce cancer in humans (remote as that may be) through an E1A-dependent mechanism.
The invention also includes vectors overexpressing ADP whose replication is restricted to specific tissues by placing expression of one or more proteins essential for replication under the control of a tissue specific promoter. A number of tissue-specific promoters have been described in the art such as the surfactant protein B promoter which is only active in cells containing the TTFI transcription factor, i.e., type II alveolar cells (Yan et al., supra) the transcriptional regulatory element described in U.S. Pat. No. 5,466,596 to Breitman et al., that directs gene expression specifically in cells of endothelial lineage; prostate specific antigen which is expressed in prostate cells (Rodriguez et al., supra); and human alpha-lactalbum gene which is expressed in breast cancer cells (Anderson et al., Gene Therapy 6:854-864, 1999). Many other tissue-specific or tissue-preferred enhancer/promoters have been reported (Miller and Whelan, Human Gene Therapy 8:803-815, 1997).
Replication of vectors according to the invention can also be controlled by placing one or more genes essential for vector replication under the control of a promoter that is activated by an exogenous inducing agent, such as metals, hormones, antibiotics, and temperature changes. Examples of such inducible promoters include but are not limited to metallothionein promoters, the glucocorticoid promoter, the tetracycline response promoter, and heat shock protein (hsp) promoters such as the hsp 65 and 70 promoters.
The invention also provides compositions comprising a recombinant vector that overexpresses ADP in an amount effective for promoting death of neoplastic cells and a method comprising administering a therapeutically effective amount of the vector to a neoplastic cell in a patient. It is believed the compositions and methods of the present invention are useful for killing neoplastic cells of any origin and include neoplastic cells comprising tumors as well as metastatic neoplastic cells.
It is also contemplated that ADP-expressing viral vectors can be administered to neoplastic cells along with a replication-defective virus that expresses an anti-cancer gene product. For example, many replication-defective E1− Ad vectors for use in cancer therapy are well characterized. A limitation of replication-defective vectors is that they only synthesize the therapeutic protein in the cell they initially infect, they cannot spread to other cells. Also, since the genome does not replicate, transcription can only occur from the input genomes, and this could be as low as one copy per cell. In contrast, the genome of replication-competent.Ad vectors are amplified by about 104 in the cell that was initially infected, providing more templates for transcription. More amplification is achieved as the vector spreads to other cells. By combining replication-defective viral vectors expressing an anti-cancer gene product with replication-competent viral vectors described herein, it is expected that the result will be template amplification and rapid spread of both vectors to surrounding cells. For example, with Ad-based vectors, the burst size for each vector should be large, ˜104 PFU/cell, so the probability of co-infection of surrounding cells by both vectors will be high. Thus, both the replication-competent and replication-defective vectors should spread simultaneously through the tumor, providing even more effective anti-cancer therapy.
Expression of the anti-cancer gene product encoded by the replication-defective vector can be under the control of either constitutive, inducible or cell-type specific promoters. The anti-cancer gene product can be any substance that promotes death of a neoplastic cell. The term “gene product” as used herein refers to any biological product or products produced as a result of the biochemical reactions that occur under the control of a gene. The gene product can be, for example, an RNA molecule, a peptide, a protein, or a product produced under the control of an enzyme or other molecule that is the initial product of the gene, i.e., a metabolic product. For example, a gene can first control the synthesis of an RNA molecule which is translated by the action of ribosomes into a prodrug converting enzyme which converts a nontoxic prodrug administered to a cancer patient to a cell-killing agent; the RNA molecule, enzyme, and the cell-killing agent generated by the enzyme are all gene products as the term is used here. Examples of anti-cancer gene products include but are not limited to cell-killing agents such as apoptosis-promoting agents and toxins; prodrug converting enzymes; angiogenesis inhibitors; and immunoregulatory molecules and antigens capable of stimulating an immune response, humoral and/or cellular, against the neoplastic cell.
Apoptosis-promoting agents include but are not limited to the pro-apoptotic members of the BCL-2 family such as BAX, BAD, BID and BIK, as well as antisense molecules which block expression of anti-apoptotic members of the BCL-2 family. Examples of immunoregulatory molecules are cytokines such as tumor necrosis factor, Fas/Apo1/CD95 ligand, tumor necrosis factor related apoptosis inducing ligand, interleukins, macrophage activating factor and interferon γ. Angiogenesis inhibitors include but are not limited to endostatin and angiostatin. Toxins include but are not limited to tumor necrosis factor, lymphotoxin, the plant toxin ricin, which is not toxic to humans due to the lack of ricin receptors in animal cells, and the toxic subunit of bacterial toxins. Examples of pro-drug converting enzymes and pro-drug combinations are described in WO 96/40238 and include: thymidine kinase and acyclovir or gancyclovir; and bacterial cytosine deaminase and 5-fluorocytosine.
The therapeutic or pharmaceutical compositions of the present invention can be administered by any suitable route known in the art including for example by direct injection into a tumor or by other injection routes such as intravenous, subcutaneous, intramuscular, transdermal, intrathecal and intracerebral. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of slow release formulation. For treating tissues in the central nervous system, administration can be by injection or infusion into the cerebrospinal fluid (CSF). When it is intended that a recombinant vector of the invention be administered to cells in the central nervous system, administration can be with one or more agents capable of promoting penetration of the vector across the blood-brain barrier. Preferably, vectors of the invention are administered with a carrier such as liposomes or polymers containing a targeting moiety to limit delivery of the vector to targeted cells. Examples of targeting moieties include but are not limited to antibodies, ligands or receptors to specific cell surface molecules.
Compositions according to the invention can be employed in the form of pharmaceutical preparations. Such preparations are made in a manner well known in the pharmaceutical art. One preferred preparation utilizes a vehicle of physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers such as physiological concentrations of other non-toxic salts, five percent aqueous glucose solution, sterile water or the like may also be used. It may also be desirable that a suitable buffer be present in the composition. Such solutions can, if desired, be lyophilized and stored in a sterile ampoule ready for reconstitution by the addition of sterile water for ready injection. The primary solvent can be aqueous or alternatively non-aqueous.
The carrier can also contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation. Similarly, the carrier may contain still other pharrnaceutically-acceptable excipients for modifying or maintaining release or absorption or penetration across the blood-brain barrier. Such excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dosage or multi-dose form or for direct infusion into the cerebrospinal fluid by continuous or periodic infusion.
It is also contemplated that certain formulations containing ADP-expressing vectors are to be administered orally. Such formulations are preferably encapsulated and formulated with suitable carriers in solid dosage forms. Some examples of suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like. The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents. The compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art. The formulations can also contain substances that diminish proteolytic degradation and promote absorption such as, for example, surface active agents.
The specific dose is calculated according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied. The dose will also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art. Such calculations can be made without undue experimentation by one skilled in the art. Exact dosages are determined in conjunction with standard dose-response studies. It will be understood that the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration. Dose administration can be repeated depending upon the pharmacokinetic parameters of the dosage formulation and the route of administration used.
The invention also contemplates passively immunizing patients who have been treated with a viral vector overexpressing ADP. Passive immunization can include administering to the patient antiserum raised against the viral vector, or gamma-globulin or vector-specific purified polyclonal or monoclonal antibodies isolated from the antiserum. Preferably, the patient is passively immunized after a time period sufficient for the viral vector to replicate in and spread through the tumor.
Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the examples.
This example illustrates the construction and characterization of the KD1 and KD3 anti-cancer vectors.
To construct KD1, the inventors deleted the entire E3 region of a unique plasmid, leaving behind only a unique PacI site for cloning. The starting plasmid was pCRII, purchased from Invitrogen, containing the Ad5 BamHIA fragment having a deletion of all the E3 genes; the E3 deletion is identical to that for KD1 and GZ3, the sequences of which are given in SEQ ID NO:1 and SEQ ID NO:4, respectively. The ADP gene from Ad5 was cloned into the PacI site, then built into the E3 region of the genome of the Ad5 E1A mutant named dl01/07. This was done by co-transfecting into human embryonic kidney 293 cells the aforementioned BamHIA fragment containing the ADP gene together with the overlapping EcoRIA restriction fragment obtained from dl01/07. Complete viral genomes are formed within the cell by overlap recombination between the Ad sequences in the BamHIA fragment in the plasmid and the EcoRIA fragment. KD3 was constructed in the same way except the E3 gene for the 12.5K protein was retained in the starting plasmid. A vector named KD2, which marginally overexpress ADP, was also prepared. Plaques of each recombinant Ad were picked, screened, purified, expanded into CsCl-banded stocks, sequenced, titered, and characterized. GZ1 and GZ3 are Ad vectors that are identical to KD1 and KD3, respectively, except that GZ1 and GZ3 have wild-type E1A sequences as found in AD5 or in the Ad5 mutant dl309. GZ1 and GZ3 were constructed as described for KD1 and KD3 except that the EcoRIA fragment of AdS was used for GZ1 and GZ3.
KD1 and KD3 were characterized in cell culture by infecting the human A549 lung carcinoma cell line with high titer (1-8×1010 plaque forming units [PFU] per ml) virus stocks of one of these recombinant vectors, or with one of the control viruses dl01/07, dl309, dl327, and Ad5 (wt). Fifty PFU per cell were used for each virus. The descriptions of these viruses as well as some other viruses used in these examples are presented in Table 1.
TABLE 1
Description of mutations in viruses:
RNA REGION
Virus
E1
VA
E3
E4
dl1101/1107
dl1101: deletion
From dl309
From dl309 deletion of Ad5 bp 28597-28602;
wild type
of Ad5 bp 569-634
deletion of Ad5
deletion-substitution Ad5 bp 3005-30750, insert 642
dl1107: deletion
bp 10594-10595
bp DNA of unknown origin
of Ad5 bp 890-928
KD1
dl1101: deletion
From dl309
deletion of Ad5 bp 27858-2760, TAA inserted;
wild type
of Ad5 bp 569-634
deletion of Ad5
deletion of Ad5 bp 27982-28134; deletion of Ad5 bp
dl1107: deletion
bp 10594-10595
28395-29397, insert CCTTAATTAAA; deletion of
of Ad5 bp 890-928
Ad5 bp 29783-30883, insert TTAATTAAGG
KD2
dl1101: deletion
From dl309
dl309 background, gp19K mutated deletion of Ad5
wild type
of Ad5 bp 569-634
deletion of Ad5
bp 28597-28602; deletion-substitution Ad5 bp 3005-
dl1107: deletion
bp 10594-10595
30750, insert 642 bp DNA of unknown origin;
of Ad5 bp 890-928
deletion of Ad5 bp 28788-28789, insert TTAATTAA
KD3
dl1101: deletion
From dl309
deletion of Ad5 bp 28598-29397; deletion of Ad5 bp
wild type
of Ad5 bp 569-634
deletion of Ad5
29783-30469
dl1107: deletion
bp 10594-10595
of Ad5 bp 890-928
GZ1
wt
wild type
deletion of Ad5 bp 27858-2760, TAA inserted;
wild type
deletion of Ad5 bp 27982-28134; deletion of Ad5 bp
28395-29397, insert CCTTAATTAAA; deletion of
Ad5 bp 29783-30883, insert TTAATTAAGG
GZ3
wild type
wild type
deletion of AD5 bp 28598-29397; deletion of Ad5 bp
wild type
29783-30469
dl1101/1107-
dl1101: deletion
From dl309
From dl309 deletion of Ad5 bp 28597-28602;
E4 promoter
SPB
of Ad5 bp 569-634
deletion of Ad5
deletion-substitution Ad5 bp 3005-30750, insert 642
deletion-
dl1107: deletion
bp 10594-10595
bp DNA of unknown origin
substitution:
of Ad5 bp 890-928
deletion of Ad5 bp
35623-35775, insert
SP-B 500 promoter
flanked by Bst1
107I sites
KD1-SPB
dl1101: deletion
From dl309
deletion of Ad5 bp 27848-2760, TAA inserted;
E4 promoter
of Ad5 bp 569-634
deletion of Ad5
deletion of Ad5 bp 27982-28134; deletion of Ad5 bp
deletion-
dl1107: deletion
bp 10594-10595
28395-29397, insert CCTTAATTAAA; deletion of
substitution:
of Ad5 bp 890-928
Ad5 bp 29783-30883, insert TTAATTAAGG
deletion of Ad5 bp
35623-35775, insert
SP-B 500 promoter
flanked by Bst1
107I sites
KD3-SPB
dl1101: deletion
From dl309
deletion of Ad5 bp 28598-29397; deletion of Ad5 bp
E4 promoter
of Ad5 bp 569-634
deletion of Ad5
29783-30469
deletion-
dl1107: deletion
bp 10594-10595
substitution:
of Ad5 bp 890-928
deletion of Ad5 bp
35623-35775, insert
SP-B 500 promoter
flanked by Bst1
107I sites
Using a polymerase chain reaction (PCR)-based protocol, an in-frame stop codon was introduced into the gene for the E3-gp19K protein in the E3 region of the Ad5 mutant dl309 (Jones and Shenk, Cell 17:683-689, 1979). The mutagenesis was conducted using a SunI-Bst1107I fragment, nucleotides 28,390 to 29,012 in the AdS genome, which was then substituted for the equivalent fragment in dl309. dl01/07 is the parent for KD1 and KD3. In turn, the AdS mutant named dl309 is the parent of dl01/07, i.e. dl309 is identical to dl01/07 except that dl309 does not have the E1A mutation. Both dl01/07 and dl309 have deletions of the genes for the E3 RIDα, RIDβ and 14.7K proteins but retain the gene for ADP. The Ad5 mutant dl327 has wild-type E1A, it lacks the gene for ADP, and its lacks all other E3 genes except the one for the 12.5K protein.
At 24 and 36 hours post-infection (h p.i.), proteins were extracted from the A549 cells and analyzed for ADP by immunoblot using a rabbit antiserum against ADP (Tollefson et al., J. Virol. 66:3633-3642, 1992). The results are shown in
The amount of ADP detected in the KD1 and KD3 infected cells is significantly higher than the amount detected in the dl309 infected cells (
This example illustrates that KD1 and KD3 lyse cells more rapidly and spread from cell-to cell faster than other adenoviruses.
The ability of KD1 and KD3 to lyse cells was examined by a trypan blue exclusion cell viability assay which was performed essentially as described by Tollefson et al., J. Virol. 70:2296-2306, 1996. In brief, A549 cells were mock-infected or infected with 20 PFU/cell of KD1, KD3, dl01/07, dl327 or dl309. At various days p.i., the number of viable cells was determined using a hemacytometer (600 to 1000 cells were counted per time point) and the results are shown in
Only 25% of the KD1-infected cells and 9% of the KD3-infected cells were alive at 5 days p.i. as compared to 44% of cells infected with dl01/07, which has the same E1A mutation as KD1 and KD3. The KD1 and KD3 vectors also lysed cells faster than dl309, which has a wild-type E1A region. When infected with dl327 (ADP−, E1A+), 94% of the cells were alive after 5 days. When cell lysis was estimated by release of lactate dehydrogenase, KD1 and KD3 once again lysed cells faster than dl01/07 and dl309, and dl327 caused little cell lysis (data not shown). Thus, ADP is required for efficient cell lysis, and over-expression of ADP increases the rate of cell lysis.
As another means to measure cell lysis and to examine virus replication in cancer cells, separate groups of A549 cells were infected with 20 PFU/cell of KD1, KD3, dl01/07, or dl309 and the amount of intracellular and extracellular virus was determined by plaque assay on A549 cells. At 2 days p.i., the total amount of virus formed in each group was similar, 2-4×108 PFU/ml, indicating that replication of all the viruses is similar. However, when the ratio of extracellular to intracellular virus was calculated, the value for KD1 and KD3 was 2-3 logs higher than for Ad5, dl309, or dl01/07 (data not shown). Thus, virus is released much more rapidly from cells infected with KD1 and KD3, which overexpress ADP, than with viruses expressing wild-type amounts of ADP.
The ability of KD1 and KD3 to spread from cell-to-cell was measured in a “cell spreading” assay. In this assay monolayers of A549 cells in a 48 well culture dish were mock-infected or infected with 10−3, 10−2, 10−1, 100, or 10 PFU/cell of dl327, dl309, Ad5, dl01/07, KD1 or KD3. At low PFU/cell, the viruses must go through two or three rounds of replication in order to infect every cell in the monolayer. At 1.0 and 10 PFU/cell, the monolayer should be destroyed by the virus that initially infected the cells. To assess the amount of spread in the monolayers by 7 days p.i., crystal violet, which stains live cells but not dead cells, was added to the monolayers. The results are shown in
Remarkably, at 7 days p.i., the monolayer was virtually eliminated by KD1 and KD3 at 10−3 PFU/cell, whereas 1.0 PFU/cell was required with dl01/07, dl309 and Ad5. This result attests to the potency of ADP in mediating cell lysis and virus spread in A549 cells. KD1 and KD3 are also more effective that dl01/07 in killing other types of human cancer cell lines (most purchased from the American Type Culture Collection [ATCC]) as determined in this cell spreading assay. KD1 and/or KD3 killed HeLa (cervical carcinoma), DU145 (prostate), and pC3 (prostate) cells at 10−2 PFU/cell, ME-180 (cervix) and Hep3B (liver) at 10−1 PFU/cell, and U118 (glioblastoma) and U373 (glioblastoma) at 10 PFU/cell. From 10- to 100-fold more dl01/07 was required to kill these cells (data not shown). These results indicate that KD1 and KD3 may be effective against many types of cancer.
An important aspect of the finding that ADP overexpressing vectors lyse cells at very low multiplicities of infection is that the multiplicity of infection in human tumors is likely to be low at sites distal to the sight of vector injection or distal to blood vessels that carry the vector to the tumor. Thus, ADP overexpressing vectors have an advantage over vectors that express less ADP or no ADP at all.
This example illustrates that KD1 and KD3 replicate poorly in non-growing non-cancerous cells. The replication phenotype of KD1 and KD3 was evaluated using “normal” HEL-299 human fibroblast cells, either growing in 10% serum or rendered quiescent using 0.1% serum. All Ads should replicate well in growing cells, but viruses with the dl01/07 E1A mutation should do poorly in quiescent cells because E1A is required to drive them out of G0. dl309, which has wild-type E1A, should replicate well in both growing and growth-arrested cells.
Cells were infected with 100 PFU/cell of KD1, KD3, dl01/07, or dl309. At different days p.i., virus was extracted and titered. In 10% serum, KD1, KD3, and dl01/07 replicated well, reaching titers of 106-107 PFU/ml, only slightly less than dl309 (
The restriction associated with the dl01/07 E1A mutation was also tested in primary human cells (purchased from Clonetics) growing as monolayers. Bronchial epithelial cells (
In summary, the above experiments demonstrate that KD1 and KD3 lyse cancer cells, spread from cell-to-cell rapidly, and replicate poorly in quiescent and non-cancerous cells. These properties should make them useful in anti-cancer therapy.
This example illustrates that KD1 and KD3 inhibit the growth of human tumors in an animal model.
We could not evaluate mouse or rat tumors in normal mice or rats because they are totally non-permissive. Human cancer cell lines growing in nude mice have been used by Onyx Pharmaceuticals (Richmond, Calif.) to evaluate the efficacy of ONYX-015, an Ad vector lacking expression of the E1B 55 kDa protein (Heise et al., Nature Med. 3:639-645, 1997). We have found that A549 cells, which were used in many of our cell culture studies, form excellent rapidly growing solid tumors when injected subcutaneously into nude mice. The average tumor reaches ca. 500 μl in four weeks, and is encapsulated, vascularized, and attached to the mouse skin (usually) or muscle.
Nude mice were inoculated into each hind flank with 2×107 A549 cells. After 1 week tumors had formed, ranging in size from about 20 μl to 50 μl. Individual tumors were injected three days later, and at subsequent weeks for 4 weeks (total of 5 injections), with 50 μl of buffer or 50 μl of buffer containing 5×107 PFU of dl309, dl01/07, KD1, KD3, or pm734.1, with a total virus dose per tumor of 3×108 PFU. The mutant pm734.1 lacks ADP activity due to two nonsense mutations in the gene for ADP, but all other Ad proteins are expected to be synthesized at wild-type levels (Tollefson et al., J. Virol. 70:2296-2306, 1996). The efficacy of each virus (or buffer) was tested on six tumors. At weekly intervals, the length (L) and width (W) of tumors were measured using a Mitutoyo digital caliper. Tumor volumes were calculated by multiplying L×W×W/2. This value was divided by the tumor volume at the time of the initial virus injection, the fold-increase in tumor growth was calculated, and the average for the six tumors was graphed.
As shown in
The finding that KD1 and KD3 are as effective as wild-type Ad (i.e. dl309) in reducing the rate of A549 tumor growth is highly significant in the context of cancer treatment, inasmuch as KD1 and KD3 are restricted to cancer cells whereas wild-type Ad does not have such a restriction.
The tumors in
We have also found that KD1 and KD3 reduce the rate of growth in nude mice of a human liver cancer cell line, Hep3B cells. These cells form rapidly growing tumors that are highly vascularized. Nude mice were inoculated into each hind flank with 1×107 of Hep3B cells. After tumors reached about 100 μl, they were injected twice per week for 3 weeks with 50 μl of buffer or 5×107 PFU of KD1, KD3, or dl309. There were typically 8-10 tumors per test virus. The tumor sizes were measured and the fold increase in size at 0 to 3.5 following the initial virus injection was graphed as described above for the A549 tumors. Tumors that received buffer alone grew 9-fold over 3 weeks and were projected to grow about 12-fold over 3.5 weeks (after 3 weeks the mice had to be sacrificed because the tumors were becoming too large) (
These results point to the potency of ADP as an anti-tumor agent when expressed in an Ad vector. It is highly probable that KD1 and KD3 will provide significant clinical benefit when used to infect tumors growing in humans.
This example illustrates the use of replication-defective Ad vectors in combination with KD1 or KD3.
It is well established that replication-competent (RC) viruses complement replication-defective (RD) mutants. That is, when the same cell is infected, the competent virus will supply the protein(s) that cannot be made from the mutant genome, and both viruses will grow. To test the ability of KD1 and KD3 to complement RD viruses, two RD vectors expressing β-galactosidase were constructed. The first, named Ad-β-gal, has a cDNA encoding β-gal under the control of the Rous Sarcoma Virus promoter substituted for the deleted E1 region. Ad-β-gal also has the E3 region deleted, including the gene for ADP. The second, named Ad-β-gal/FasL is identical to Ad-β-gal, except that it also expresses murine FasL from the human cytomegalovirus promoter/enhancer. These vectors were constructed by overlap recombination in human 293 cells that constitutively express the Ad E1A and E1B genes and complement replication of the E1-minus vectors.
These RD vectors should infect and express β-gal in A549 cells, but should not replicate because the E1A proteins are lacking. However, the vectors should replicate when cells are co-infected with RC Ads. To prove this, A549 cells were infected with 10 PFU/cell of Ad-β-gal alone, or with 10 PFU/cell of Ad-β-gal plus 10 PFU/cell of KD1, KD3, dl01/07, dl309, or dl327. At 2 days p.i., virus was extracted and Ad-β-gal titers determined by β-gal expression in A549 cells. The yields are shown in Table 2 below.
TABLE 2
Yield
Virus
(blue plaques per ml)
Ad-β-gal
1 × 102
Ad-β-gal + KD1
2 × 105
Ad-β-gal + KD3
3 × 105
Ad-β-gal + dl01/07
4 × 104
Ad-β-gal + dl309
3 × 105
Ad-β-gal + dl327
3.0 × 105
The data in Table 2 indicate that the complementing viruses increased the yield of Ad-β-gal by about 103.
A key feature of KD1 and KD3 is that they spread from cell-to-cell faster than other Ads. Accordingly, they should complement the spread of Ad-β-gal. To test this, an infectious center assay was conducted. A549 cells were infected with Ad-β-gal plus KD1, KD3, or dl01/07. After 2 h, cells were collected, diluted, and seeded onto monolayers of fresh A549 cells. After 4 days, the cells were stained with X-gal and the results are shown in
With Ad-β-gal alone, only the originally infected cell (before seeding) should be stained, and the vector should not spread to other cells on the seeded monolayer. This was indeed the case. In monolayers seeded with A549 cells infected with Ad-β-gal alone (dish shown in the top left of
It is expected that KD1 and KD3 will also complement and enhance the spread of RD vectors expressing anti-cancer therapeutic gene products, and this expectation can be readily verified using the Ad-β-gal/FasL in replication and infectious center assays as described above.
KD1 and KD3 not only complement the replication of RD vectors in cell culture, they also do so in Hep3B tumors growing in the hind flanks of nude mice. The RD vector used was AdLuc, an Ad that lacks the E1 and E3 regions, and has inserted into the E1 region an expression cassette where the firefly luciferase gene is expressed from the Rous Sarcoma Virus promoter (Harrod et al., Human Gene Therapy 9:1885-1898, 1998). The Hep3B tumors were injected with 1×107 PFU of AdLuc plus buffer, or 1×107 PFU of AdLuc plus 5×107 PFU of KD1, KD3, dl01/07, or dl309. After 2 weeks, mice were sacrificed and tumors excised. Proteins were extracted from the tumors and luciferase activity determined using a luminometer. The luciferase counts per tumor were 6,800 for AdLuc plus buffer, 113,500 for KD1, and 146,900 for KD3 (
In summary, infecting a tumor with a replication-competent ADP-overexpressing vector according to the invention together with a RD vector expressing an anti-cancer gene product should greatly increase the amount of anti-cancer protein synthesized in the tumor thereby increasing the ability of the replication-defective vector to promote destruction of the tumor.
This example illustrates the construction and characterization of a recombinant Ad vector according to the invention which is replication-restricted to cancerous type II alveolar cells.
As demonstrated above, the dl01/07 mutation in KD1 and KD3 limits growth of these vectors to cancer cells. To further restrict their replication phenotype, the E4 promoter in each virus was deleted and replaced by the surfactant protein B (SPB) promoter to produce vectors named KD1-SPB (SEQ ID NO:14), KD3-SPB (SEQ ID NO:15), and dl01/07-SPB (SEQ ID NO:16). The SPB promoter is only active in cells containing the TTF1 transcription factor, which has thus far been found primarily in type II alveolar cells of the human lung (Lazzaro et al., Development 113:1093-1104, 1991). Thus, KD1-SPB, KD3-SPB, and dl01/07-SPB should be severely restricted to cancerous type II alveolar cells of the human lung. Many lung cancers are of this type.
The KD1-SPB and KD3-SPB vectors were prepared as follows. The E4 promoter is located at the right end of the Ad genome (
The A549-TTF1 cell line was developed in order to test the prediction that replication of dl01/07-SPB, KD1-SPB, and KD3-SPB would be restricted to cancerous cells expressing the TTF1 transcription factor. These cells were co-transfected with two plasmids, one in which TTF1 is expressed from the CMV promoter, and the other coding for resistance to neomycin Resistant clones were isolated and shown to express TTF1 activity as determined by transient transfection with a plasmid expressing chloramphenicol acetyltransferase from the TFT1-requiring surfactant protein C promoter.
KD1-SPB and KD1 were subjected to a standard plaque development assay on A549-TTF1 cells and parental A549 cells. The results are shown in
The restriction of KD1-SPB to cells containing TTF1 was further examined in a cell spread assay using H441 cells, a TTF1-expressing human pulmonary adenocarcinoma cell line (Yan et al., supra), and Hep3B cells, a liver cancer cell line not expected to express TTF1. Culture dish wells containing H441 or Hep3B cells were infected with KD1-SPB or KD1 at multiplicities ranging from 10 to 10−4 PFU/cell. The H441 and Hep3B cells were stained with crystal violet at 5 days and 8 days p.i., respectively. KD1-SPB and KD1 grew and spread equally well on H441 cells, causing destruction of the monolayer at 10−1 PFU per cell (
In summary, this example demonstrates that a replication-competent Ad, which replicates well on cells expressing the appropriate transcription factor, can be constructed with a tissue-specific promoter substituted in place of the E4 promoter. This methodology should be applicable to many other tissue specific and cell type specific promoters. One possibility would be a liver-specific promoter. Another possibility would be to use the E2F promoter, or another promoter with E2F sites, inasmuch as that promoter would be active only in cells such as cancer cells that have free E2F. A third possibility would be to use a regulatable promoter, e.g. the synthetic tetracycline response promoter (Massie et al., J. Virol. 72:2289-2296, 1998), where the activity of the promoter is controlled by the level of tetracycline or a tetracyclin analog in the patient.
This example illustrates the construction and characterization of vectors which overexpress ADP and are not replication restricted.
As demonstrated above, the dl01/07 E1A mutation in KD1 and KD3 is attenuating, inhibiting growth in non-dividing and even in dividing primary human epithelial and endothelial cells. Ads with this mutation are able to replicate well in dividing cancer cells. However, replication of such E1A mutants is not as efficient as, e.g. dl309 which has a wild-type E1A gene. For instance, the rate of replication of dl01/07, as determined by the rate at which plaques develop, is reduced such that dl01/07 plaques appear one day later than those of dl309 (data not shown). This delay is due in part to a delay in expression of Ad late genes (see
In light of these considerations, vectors named GZ1 and GZ3 have been constructed that are identical to KD1 and KD3, respectively, except they have a wild-type E1A region. These vectors were constructed by overlap recombination in A549 cells. The left hand fragment contained the wild-type E1A region of Ad5, and the right end fragment contained the E3 modifications of KD1 or KD3. Plaques were picked, analyzed for the expected genotype, plaque-purified, and expanded into CsCl-banded stocks. The titers of these stocks on A549 cells were 2.9×1010 PFU/ml for GZ1 and 1.6×1011 PFU/ml for GZ3. Thus, these vectors can be grown into high titer stocks comparable to wild-type Ad. The GZ1 and GZ3 plaques are larger and appear much sooner than the plaques for dl309. Large rapidly-appearing plaques reflect the ability of Ad to lyse cells and spread from cell-to-cell (Tollefson et al., J. Virol. 70:2296-2306, 1996; Tollefson et al., Virology 220:152-162, 1996), and this property, as discussed, is due to the function of ADP.
The rate of plaque appearance can be quantitated in a plaque development assay (Tollefson et al., supra). Here, a typical plaque assay is performed, and the plaques observed on subsequent days of the assay are calculated as a percentage of the number of plaques observed at the end of the plaque assay. As shown in
GZ1 and GZ3 lyse cells and spread from cell to cell much more effectively than dl309. At 6 days p.i. of A549 cells, approximately as much monolayer destruction was observed with GZ1 and GZ3 at 10−3 PFU per cell as was observed with dl309 at 10−1 PFU per cell (
In theory, GZ1 and GZ3 should be able to replicate not only in tumor cells but also in normal cells. Although they can replicate in normal cells, it is quite possible that GZ1 and GZ3 may be useful as anti-cancer vectors. First, GZ1 and GZ3 could be injected directly into the tumor. Many tumors are self-contained (encapsulated) except for the blood supply. The physical barriers of the tumor could minimize dissemination of the virus to other tissues. Second, Ads are in general quite benign. Most infections of Ad5 are in infants and result in mild or asymptomatic disease, and are held in check by strong humoral and cellular immunity. Anti-Ad immunity appears to be life-long. GZ1 and GZ3 could be used only in patients who have an intact immune system, and perhaps also with pre-existing anti-Ad immunity. Further, patients could be passively immunized against Ad, using gamma-globulin or even specific purified anti-Ad neutralizing antibodies. Third, considering that Ad5 is a respiratory virus which most efficiently infects lung epithelial cells displaying the specific Ad5 receptor (named CAR) as well as specific integrins (e.g. av b5), replication-competent vectors derived from AdS may not spread efficiently in many non-cancer tissues of the body. In addition, it is believed that versions of GZ1 and GZ3 can be constructed that have the E4 promoter substituted with a tumor-specific, tissue-specific, cell-specific, or synthetic promoter. Such vectors would have the positive features associated with wild-type E1A and ADP, and yet be replication-restricted to tumor tissue and/or to particular cell types.
This example illustrates that the combination of KD1, KD3, GZ1, or GZ3 with radiation is more effective in destroying A549 cells, growing in culture or growing as tumors in nude mice, than the vectors alone or radiation alone.
This was shown in a cell spread assay. A549 cells growing in three 48 well culture dishes were mock-infected or infected with different viruses at multiplicites of infection ranging from 10 to 10−4 pFU per cell as indicated in
With the dish that was infected then radiated with 600 cGy there was markedly increased cell killing and virus spread as compared to the non-radiated cells (compare the bottom panel of
The amount of cell destruction was quantitated by extracting the crystal violet from the cells with 33% acetic acid, then measuring the absorbance at 490 nm (data not shown). The absorbance with non-radiated mock-infected cells was set at 100% cell viability. With mock-infected cells that received 600 cGy there was a 15% loss in viability (i.e. 15% less crystal violet was extracted). With KD1 at 10−3 PFU per cell, the non-radiated cells were 80% viable whereas the cells receiving 600 cGy of radiation were only about 30% viable. Similar differences in viability between radiated and non-radiated cells were seen with KD3, GZ1, and GZ3. These results argue that the combination of radiation plus vector has a syngergistic effect on cell lysis and vector spread, rather than an additive effect. If the effect were only additive, then with the KD1 samples at 10−3 PFU per cell, the cell viability should have been 65% (15% reduction in viability due to radiation alone, 20% reduction due to KD1 alone). In fact, the cell viability was 30% rather than 65%.
As mentioned, approximately as much cell lysis and virus spread were observed with 600 cGy as with 2000 cGy. To determine the optimal dose of radiation to synergize with the vectors, an experiment similar to the one described above was conducted with mock-, dl01/07-, KD1-, KD3-, dl309, GZ1-, or GZ3-infected A549 cells. The 48 well plates received 0, 150, 300, or 600 cGy of radiation at 24 h p.i. Cells were stained with crystal violet. The results with cells receiving 0 versus 600 cGy of radiation were similar to those in
The combination of KD3 or GZ3 with radiation was also examined in the A549 tumor-nude mouse model (see Example 4). A549 cells were injected into the hind flanks of nude mice, and tumors were allowed to form. When tumors reached approximately 50-μl, they were injected with buffer or with 5×108 PFU of KD3 or GZ3. Eight to ten tumors were injected per test condition. At 1 day p.i., half the mice received 600 cGy of whole body radiation. Tumor size was measured over time, and was plotted as a fold-increase in tumor size versus days p.i. (as described in Example 4). As shown in
The mechanism by which radiation causes the ADP overexpressing vectors to lyse cells and spread from cell-to-cell more effectively is not understood. Radiation is expected to induce cellular DNA repair mechanisms, and that may allow for more efficient synthesis of Ad DNA. Radiation may enhance the function of ADP. ADP probably functions by interacting with one or more cellular proteins, and radiation may affect this protein(s) such that ADP functions more efficiently.
It is believed that KD1, KD3, GZ1, or GZ3, or any other replication-competent Ad vector, when used in combination with radiation, will be more effective than vector alone or radiation alone in providing clinical benefit to patients with cancer. The vectors should allow more tumor destruction with a given amount of radiation. Stated another way, radiation should cause more tumor destruction with a given amount of vector. These vectors should also allow the radiation oncologist to use less radiation to achieve the same amount of tumor destruction. Less radiation would reduce the side effects of the radiation.
It is also believed that a cocktail of vectors when used in combination with radiation will be more effective than the cocktail alone or radiation alone. The cocktail could consist of ADP producing vectors plus one or more replication defective vectors expressing an anticancer therapeutic protein (see Example 5).
This example illustrates a structure-function analysis of adenovirus death protein.
ADP is an 11.6 kDa N-linked O-linked integral membrane glycoprotein that localizes to the inner nuclear membrane (NM) (Scaria et al., Virology 191:743-753). As illustrated in
The structure of ADP in rec700 and in each deletion mutant is schematically illustrated in
The ability of these mutants to promote cell death was monitored by trypan blue exclusion, plaque development, and lactate dehydrogenase release assays (Tollefson et al., J. Virol. 70:2296-2306, 1996). The trypan blue results in
The effects of these mutations on the intracellular localization of ADP are extremely interesting. When examined by immunofluorescence (IF) at 33 h p.i. (data not shown), ADP from rec700 (WT) localized crisply to the NM; localization to the Golgi was also apparent. With dl714 (Δ71-94) and dl715 (Δ76-89), ADP localized to all membranes, i.e. the ER, Golgi, plasma membrane, and NM. This was even more apparent at 45 h p.i. (data not shown) Thus, aa 71-94 appear to include a signal that directs ADP specifically to the NM. ADP is very likely sorted from the trans-Golgi network (TGN) to the NM, so this putative signal in ADP probably functions in this sorting pathway. ADP from dl717 (Δ81-88) is intriguing: it localized to the NM and Golgi, but in many cells “dots” and circular structures were observed. Again, this was more apparent at 45 h p.i. when these structures were the prominent feature. dl717-infected cells have not begun to die at 45 h p.i., so these structures are not cellular remnants. The intriguing possibility is that these structures are membrane vesicles that have pinched off from the TGN but are defective in targeting to and/or fusing with the NM.
With dl738 (Δ46-60 in the SA domain), ADP aggregated in the cytoplasm. This again indicates that aa 46-60 include the SA sequence. With pm734.4 (M56), ADP localized primarily to the NM. As discussed above, the pm734.4 ADP has exclusively high-mannose N-linked oligosaccharides, indicating that it never leaves the ER. Perhaps the putative NM-localization signal in the C-terminal region of the pm734.4 ADP targets ADP to the NM by lateral diffusion from the ER (which is continuous with the outer and inner NM).
With dl737 (Δ29-45), ADP localized to the NM. ADP from pm734 (Δ1-40), pm734.7 (N14) (N-linked glycosylation cannot occur), and dl735 (Δ4-11; the O-glycosylation sites are deleted) localized much more prominently to the Golgi than the NM. ADP from dl735.1 (Δ18-22) and dl736.1 (Δ11-26) also localized much more strongly to the Golgi than the NM. Thus, residues 1-26 and/or glycosylation appear to be required for efficient transport of ADP from the Golgi/TGN to the NM.
In summary, aa 41-59 include the SA domain, Met56 in the SA domain is required for exit from the ER, aa 1-26 are required for efficient exit from the Golgi, and aa 76-94 are required to target ADP specifically to the NM. With respect to promoting cell death, the essential regions are aa 1-26, the SA domain (ADP does not enter membranes), Met56 in the SA domain, and the BP domain (aa 63-70). It is not clear whether the defective death-promoting phenotype of pm734 (Δ1-40), dl735 (Δ4-11), dl735.1 (Δ18-22), dl736.1 (Δ11-26), and pm734.7 (N14) is due to lack of sequences (or oligosaccharides) that promote death or to much slower exit of ADP from the Golgi to the NM. dl714 (Δ71-94) and dl715 (Δ76-89) express a wild-type phenotype for promoting death even though they are defective in localizing specifically to the NM; this is probably because sufficient ADP still enters the NM to promote death. Even though the deletion in d717 (Δ81-88) lies within the deletions in dl715 (Δ76-89) and dl714 (Δ71-94), the dl77 ADP is only about 15% as efficient as rec700 (WT), dl175 and dl174 in promoting death. This may be because the dl717 ADP tends to remain in vesicles rather than localizing to the NM. Altogether, these data indicate that ADP must localize to the NM in order to promote cell death.
In view of the above, it will be seen that the several advantages of the invention are achieved and other advantageous results attained.
As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.
All references cited in this specification, including patents and patent applications, are hereby incorporated by reference. The discussion of references herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinency of the cited references.
Wold, William S. M., Toth, Karoly, Doronin, Konstantin, Tollefson, Ann E.
| Patent | Priority | Assignee | Title |
| Patent | Priority | Assignee | Title |
| 5677178, | Feb 16 1993 | Onyx Pharmaceuticals, Inc. | Cytopathic viruses for therapy and prophylaxis of neoplasia |
| 5846945, | Feb 16 1993 | Onyx Pharmaceuticals, Inc. | Cytopathic viruses for therapy and prophylaxis of neoplasia |
| 6066624, | Aug 26 1993 | Baylor College of Medicine | Gene therapy for solid tumors using adenoviral vectors comprising suicide genes and cytokine genes |
| 6197293, | Mar 03 1997 | CG ONCOLOGY, INC | Adenovirus vectors specific for cells expressing androgen receptor and methods of use thereof |
| 6254862, | Mar 03 1997 | Cell Genesys, Inc | Adenovirus vectors specific for cells expressing alpha-fetoprotein and methods of use thereof |
| 6627190, | Jul 12 1999 | SAINT LOUIS UNIVERSITY | Recombinant adenovirus vectors that are replication-competent in tert-expressing cells |
| 7589069, | Jul 12 1999 | SAINT LOUIS UNIVERSITY | Replication-competent anti-cancer vectors |
| 7608255, | Jul 12 1999 | SAINT LOUIS UNIVERSITY | Replication-competent anti-cancer vectors |
| WO9839465, |
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